A vaccine may comprise an adjuvant, a diluent, a preservative, a stabilizing agent and a buffer together with an antigenic substance. In particular, an adjuvant is a substance that is administered together with an antigen and may enhance an immunological response to the antigen administered. An adjuvant may act in various ways depending on its kind, e.g. (1) it adsorbs an antigen to accelerate its intake into antigen-presenting cells; (2) it retains an antigen in a local area for a long period of time to release the antigen gradually so as to prolong antigenic stimulation; or (3) it directly activates immunocompetent cells. Thus, an adjuvant is very advantageous in reducing a dose and the number of administration of a vaccine or an amount of an antigen contained in a vaccine. Therefore, there have hitherto been made a variety of studies concerning an adjuvant that may enhance the action of a vaccine. However, adjuvants that have actually been used in medical settings are quite few.
A typical adjuvant includes aluminum hydroxide (hereinafter referred to as “Alum adjuvant”) that has been used in various vaccines. Alum adjuvant however is not an ideal adjuvant from the viewpoint of inconvenience that it can hardly be mixed uniformly with an antigen due to its insolubility and that it cannot easily be combined with a device for nasal or transdermal administration. Other than Alum adjuvant, there are adjuvants using squalene or MPL (monophosphoryl lipid). However, although they may exert a strong adjuvant activity, they exhibit severe adverse side effects and are disadvantageously insoluble in water. Thus, in medical settings, there is a desire for development of an adjuvant that may induce an elevated immunological reaction in human body, may exert few adverse side effects, and may exhibit enhanced convenience.
On the other hand, citrulline, a kind of an amino acid, is one of compounds that constitute the urea cycle and is prevalent among animals, in particular, mammals [Chemical formula: C6H13N3O3, 2-amino-5-(carbamoylamino)-pentanoic acid, Molecular weight: 175.2 g/mol]. Citrulline was found from watermelon in Japan in 1930 and is named after Citrullus vulgaris, a scientific name of watermelon.
Although citrulline is not an amino acid constituting a protein in the living body, it is one of intermediate compounds of the urea cycle. Citrulline is produced from arginine together with nitrogen oxide (NO) known as a substance with a vasodilating activity and is condensed with aspartic acid to regenerate arginine. Citrulline is known to exhibit useful activities such as acceleration of ammonium metabolism (Non-patent reference 1), improvement of blood flow due to vasodilation (Non-patent reference 2), decrease in blood pressure (Non-patent reference 3), neurotransmission (Non-patent reference 4) and elimination of active oxygen (Patent reference 1).
Therefore, citrulline has been approved as food material such as supplement in Japan in 2007. Citrulline has been used overseas earlier. Citrulline is commercially available as supplements for improvement of blood flow, prevention of arteriosclerosis and energy enhancement in the U.S. and citrulline-malate is commercially available as a medicament for recovery from fatigue in Europe.
Notwithstanding that a variety of activities of citrulline have been reported, it is not known that citrulline has an adjuvant activity. A vaccine comprising citrulline and an antigen as well as an adjuvant comprising citrulline have not been reported. Also, there is no report as to finding that citrulline and an antioxidant may be used together as an adjuvant.    Patent reference 1: Japanese Patent Publication No. 2002-226370    Patent reference 2: Japanese Patent Publication No. 53-075387    Patent reference 3: Japanese Patent Publication No. 63-068091    Patent reference 4: WO 2011024748    Patent reference 5: WO 2008133208    Non-patent reference 1: Cell Biochemistry and Function, “Effect of arginine, ornithine and citrulline supplementation upon performance and metabolism of trained rats”, 2003, Vol. 21, p. 85-91    Non-patent reference 2: European Journal of Pharmacology, “L-Citrulline mediated relaxation in the control and lipopolysaccharide-treated rat aortic rings”, 2001, Vol. 431, p. 61-69    Non-patent reference 3: Journal of Clinical Investigation, “L-arginine abrogates salt-sensitive hypertension in Dahl/Rapp rats”, 1991, Vol. 88, p. 1559-1567    Non-patent reference 4: Gastroenterology, “L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation”, 1997, Vol. 112, p. 1250-1259    Non-patent reference 5: The Journal of Biological Chemistry, “A SIMPLE SYNTHESIS OF dl-CITRULLINE”, 1938, Vol. 122, p. 477-p 484    Non-patent reference 6: The Journal of Organic Chemistry, “THE SYNTHESIS OF d,l-CITRULLINE FROM NON-BIOLOGICAL PRECURSORS”, 1941, Vol. 6, p. 410-416